Vertebrate Blood Research Articles

Entomological risk factors for potential transmission of Rift Valley fever virus around concentrations of livestock in Colorado.

Rift Valley fever virus (RVFV) poses a major threat of introduction to several continents, including North America. Such an introduction could cause significant losses to the livestock industry, in addition to substantial human morbidity and mortality. Because of the opportunistic blood host selection of Culex tarsalis mosquitoes, we hypothesized that this species could be an important bridge vector of RVFV near feedlots in the event of an introduction. We investigated the mosquito community composition at livestock feedlots and surrounding natural and residential areas to determine differences in mosquito relative abundance and blood feeding patterns attributed to cattle feeding operations. DNA extracted from abdomens of blood-fed mosquitoes were sequenced to determine host identity. Multivariate regression analyses revealed differences between mosquito community assemblages at feedlots and non-feedlot sites (p<0.05), with this effect driven largely by differential abundances of Aedes vexans (padj <0.05). Mosquito diversity was lower on feedlots than surrounding areas for three out of four feedlots. Culex tarsalis was abundant at both feedlots and nearby sites. Diverse vertebrate blood meals were detected in Cx. tarsalis at non-feedlot sites, with a shift towards feeding on cattle at feedlots. These data support a potential for Cx. tarsalis to serve as a bridge vector of RVFV between livestock and humans in Colorado.

The molecular and cellular basis of olfactory response to tsetse fly attractants.

Dipteran or “true” flies occupy nearly every terrestrial habitat, and have evolved to feed upon a wide variety of sources including fruit, pollen, decomposing animal matter, and even vertebrate blood. Here we analyze the molecular, genetic and cellular basis of odor response in the tsetse fly Glossina morsitans, which feeds on the blood of humans and their livestock, and is a vector of deadly trypanosomes. The G. morsitans antenna contains specialized subtypes of sensilla, some of which line a sensory pit not found in the fruit fly Drosophila. We characterize distinct patterns of G. morsitans Odor receptor (GmmOr) gene expression in the antenna. We devise a new version of the “empty neuron” heterologous expression system, and use it to functionally express several GmmOrs in a mutant olfactory receptor neuron (ORN) of Drosophila. GmmOr35 responds to 1-hexen-3-ol, an odorant found in human emanations, and also alpha-pinene, a compound produced by malarial parasites. Another receptor, GmmOr9, which is expressed in the sensory pit, responds to acetone, 2-butanone and 2-propanol. We confirm by electrophysiological recording that neurons of the sensory pit respond to these odorants. Acetone and 2-butanone are strong attractants long used in the field to trap tsetse. We find that 2-propanol is also an attractant for both G. morsitans and the related species G. fuscipes, a major vector of African sleeping sickness. The results identify 2-propanol as a candidate for an environmentally friendly and practical tsetse attractant. Taken together, this work characterizes the olfactory system of a highly distinct kind of fly, and it provides an approach to identifying new agents for controlling the fly and the devastating diseases that it carries.

Nucleation and Formation of a Primary Clot in Insect Blood

Blood clotting at wound sites is critical for preventing blood loss and invasion by microorganisms in multicellular animals, especially small insects vulnerable to dehydration. The mechanistic reaction of the clot is the first step in providing scaffolding for the formation of new epithelial and cuticular tissue. The clot, therefore, requires special materials properties. We have developed and used nano-rheological magnetic rotational spectroscopy with nanorods to quantitatively study nucleation of cell aggregates that occurs within fractions of a second. Using larvae of Manduca sexta, we discovered that clot nucleation is a two-step process whereby cell aggregation is the time-limiting step followed by rigidification of the aggregate. Clot nucleation and transformation of viscous blood into a visco-elastic aggregate happens in a few minutes, which is hundreds of times faster than wound plugging and scab formation. This discovery sets a time scale for insect clotting phenomena, establishing a materials metric for the kinetics of biochemical reaction cascades. Combined with biochemical and biomolecular studies, these discoveries can help design fast-working thickeners for vertebrate blood, including human blood, based on clotting principles of insect blood.

Magic roundabout is an endothelial-specific ohnolog of ROBO1 which neo-functionalized to an essential new role in angiogenesis.

BackgroundMagic roundabout (ROBO4) is an unusual endothelial-specific paralog of the family of neuronally-expressed axon guidance receptors called roundabouts. Endothelial cells (ECs), whose uninterrupted sheet delimits the lumen of all vertebrate blood vessels and which are absent from invertebrate species, are a vertebrate-specific evolutionary novelty.ResultsHerein, the evolutionary mechanism of the duplication, retention and divergence of ROBO4 was investigated for the first time. Phylogenetic analyses carried out suggested that ROBO4 is a fast-evolving paralog of ROBO1 formed at the base of vertebrates. The ancestral expression pattern was neuronal. ROBO4 dramatically shifted its expression and became exceptionally specific to ECs. The data-mining of FANTOM5 and ENCODE reveals that ROBO4’s endothelial expression arises from a single transcription start site (TSS), conserved in mouse, controlled by a proximal promoter with a complex architecture suggestive of regulatory neo-functionalization. (An analysis of promoter probabilities suggested the architecture was not due to a chance arrangement of TFBSes). Further evidence for the neo-functionalization of ROBO4 comes from the analysis of its protein interactions, the rates of protein evolution, and of positively selected sites.ConclusionsThe neo-functionalization model explains why ROBO4 protein acquired new context-specific biological functions in the control of angiogenesis. This endothelial-specific roundabout receptor is an illustrative example of the emergence of an essential vertebrate molecular novelty and an endothelial-specific signaling sub-network through 2R-WGD. The emergence of novel cell types, such as ECs, might be a neglected evolutionary force contributing to the high rate of retention of duplicates post-2R-WGD. Crucially, expression neo-functionalization to evolutionarily novel sites of expression conceptually extends the classical model of neo-functionalization.

Patterns of soft tissue and cellular preservation in relation to fossil bone tissue structure and overburden depth at the Standing Rock Hadrosaur Site, Maastrichtian Hell Creek Formation, South Dakota, USA

Recovery of soft tissues and cells from fossil bones is becoming increasingly common, with structures morphologically consistent with vertebrate osteocytes, blood vessels, fibrous/collagenous matrix, and potential intravascular contents now recognized from specimens dating back to the Permian. However, it largely remains unclear how bone tissue structure, early diagenetic regimes, and many other taphonomic variables influence or control the preservation potential of soft tissues in vertebrate fossils. To explore the influence of a few of these factors, we tested a suite of fossils from the Standing Rock Hadrosaur Site, a vast Edmontosaurus annectens bonebed in the Maastrichtian Hell Creek Formation of South Dakota, for preservation of cellular and tissue components. Demineralization of bone samples from each specimen yielded abundant microstructures morphologically consistent with vertebrate osteocytes, blood vessels, and collagenous matrix. This includes the first recovery of osteocytes and vessels from a fossil vertebral centrum and ossified tendons. Perhaps surprisingly, no correlation was found between soft tissue/cellular recovery and either bone tissue structure type (cortical vs. cancellous) or overburden depth at the time of discovery. A traditional taphonomic survey of the site, conducted in parallel and reported previously, affords a clear and detailed history of these remains, both pre- and postburial. Cumulative taphonomic evidence indicates the Edmontosaurus individuals died in a mass mortality event and their disarticulated remains were buried rapidly in a shallow floodplain pond during a crevasse splay event. Oxygenated flood waters and/or groundwater oxidized initially sideritic concretions to goethite during early diagenesis, facilitating rapid cementation of portions of the sediment that likely aided stabilization of soft tissues by shielding regions of the bones from prolonged exposure to pore fluids. Our findings support cancellous bone as a viable target for cellular analyses, corroborate previous propositions that iron-rich environments and rapid burial facilitate soft tissue preservation, and provide new details into early diagenetic environments conducive to such preservation.

In silico Prediction of Inhibitory Constant of Thrombin Inhibitors Using Machine Learning.

Thrombin is the central protease of the vertebrate blood coagulation cascade, which is closely related to cardiovascular diseases. The inhibitory constant Ki is the most significant property of thrombin inhibitors. This study was carried out to predict Ki values of thrombin inhibitors based on a large data set by using machine learning methods. Taking advantage of finding non-intuitive regularities on high-dimensional datasets, machine learning can be used to build effective predictive models. A total of 6554 descriptors for each compound were collected and an efficient descriptor selection method was chosen to find the appropriate descriptors. Four different methods including multiple linear regression (MLR), K Nearest Neighbors (KNN), Gradient Boosting Regression Tree (GBRT) and Support Vector Machine (SVM) were implemented to build prediction models with these selected descriptors. The SVM model was the best one among these methods with R2=0.84, MSE=0.55 for the training set and R2=0.83, MSE=0.56 for the test set. Several validation methods such as yrandomization test and applicability domain evaluation, were adopted to assess the robustness and generalization ability of the model. The final model shows excellent stability and predictive ability and can be employed for rapid estimation of the inhibitory constant, which is full of help for designing novel thrombin inhibitors.

The conserved metalloprotease invadolysin is present in invertebrate haemolymph and vertebrate blood.

ABSTRACTWe identified invadolysin, a novel essential metalloprotease, for functions in chromosome structure, cell proliferation and migration. Invadolysin also plays an important metabolic role in insulin signalling and is the only protease known to localise to lipid droplets, the main lipid storage organelle in the cell. In silico examination of the protein sequence of invadolysin predicts not only protease and lipase catalytic motifs, but also post-translational modifications and the secretion of invadolysin. Here we show that the protease motif of invadolysin is important for its role in lipid accumulation, but not in glycogen accumulation. The lipase motif does not appear to be functionally important for the accumulation of lipids or glycogen. Post-translational modifications likely contribute to modulating the level, localisation or activity of invadolysin. We identified a secreted form of invadolysin in the soluble fraction of invertebrate hemolymph (where we observe sexually dimorphic forms) and also vertebrate plasma, including in the extracellular vesicle fraction. Biochemical analysis for various post-translational modifications demonstrated that secreted invadolysin is both N- and O-glycosylated, but not apparently GPI-linked. The discovery of invadolysin in the extracellular milieu suggests a role for invadolysin in normal organismal physiology.

Second-Best Is Better Than Nothing: Cockroaches as a Viable Food Source for the Kissing Bug Triatoma recurva (Hemiptera: Reduviidae).

Kissing bugs in the genus Triatoma are obligate blood feeders that feed mainly on vertebrate blood and have lost the predatory lifestyle found in other reduviid bugs. They occasionally also feed on the hemolymph of arthropods, especially during the first and second instar stages. The largest kissing bug species in the United States, Triatoma recurva (Stål) (Hemiptera: Reduviidae), is poorly known and was chosen to investigate its ability to feed and develop on a diet of cockroach hemolymph. Molting from first instar individuals to second instars readily occurred at approximately the same rate reported for the species feeding on mammalian blood. Subsequent instars also fed on and survived on cockroach hemolymph with some individuals maturing to adults. In the larger instars, development time and survival rates were reduced relative to the results reported in the literature for mammalian-blood-fed individuals. Two other species of kissing bugs, Triatoma protracta (Uhler) and T. rubida (Uhler) failed to survive on cockroach hemolymph with most individuals failing to molt from the first instar stage. Although T. recurva does not thrive on a diet limited to hemolymph of cockroaches, it appears to be an unusual species in which cockroaches might be a primary source of nutrition for smaller individuals and are a viable exclusive source of nutrition for all immatures. At a minimum during times of limited availability of vertebrate blood sources, the presence of cockroaches enhances survival opportunities. Efforts to control populations of this kissing bug species likely will be improved with additional control of cockroach populations in the environment.

Fresh-blood-free diet for rearing malaria mosquito vectors

Mosquito breeding depends on the supply of fresh vertebrate blood, a major bottleneck for large-scale production of Anopheles spp. Feeding alternatives to fresh blood are thus a priority for research, outdoor large-cage trials and control interventions. Several artificial meal compositions were tested and Anopheles oogenesis, egg laying and development into the next generation of adult mosquitoes were followed. We identified blood-substitute-diets that supported ovarian development, egg maturation and fertility as well as, low progeny larval mortality, and normal development of offspring into adult mosquitoes. The formulated diet is an effective artificial meal, free of fresh blood that mimics a vertebrate blood meal and represents an important advance for the sustainability of Anopheles mosquito rearing in captivity.

Uncovering vector, parasite, blood meal and microbiome patterns from mixed-DNA specimens of the Chagas disease vector Triatoma dimidiata.

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding.

Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector Aedes aegypti

Disease-transmitting female mosquitoes require a vertebrate blood meal to produce their eggs. An obligatory hematophagous lifestyle, rapid reproduction, and existence of a large number of transmittable diseases make mosquitoes the world's deadliest animals. Attaining optimal body size and nutritional status is critical for mosquitoes to become reproductively competent and effective disease vectors. We report that blood feeding boosts serotonin concentration and elevates the serotonin receptor Aa5HT2B (Aedes aegypti 5-hydroxytryptamine receptor, type 2B) transcript level in the fat-body, an insect analog of the vertebrate liver and adipose tissue. Aa5HT2B gene disruption using the CRISPR-Cas9 gene-editing approach led to a decreased body size, postponed development, shortened lifespan, retarded ovarian growth, and dramatically diminished lipid accumulation. Expression of the insulin-like peptide (ILP) genes ilp2 and ilp6 was down-regulated while that of ilp5 and ilp4 was up-regulated in response to Aa5HT2B disruption. CRISPR-Cas9 disruption of ilp2 or ilp6 resulted in adverse phenotypes similar to those of Aa5HT2B disruption, while ilp5 CRISPR-Cas9 disruption had exactly the opposite effect on growth and metabolism, with significantly increased body size and elevated lipid stores. Simultaneous CRISPR-Cas9 disruption of Aa5HT2B and ilp5 rescued these phenotypic manifestations. Aa5HT2B RNAi silencing rendered ilp6 insensitive to serotonin treatment in the cultured fat-body, suggesting a regulatory link between Aa5HT2B and ILP6. Moreover, CRISPR-Cas9 ilp6 disruption affects expression of ilp-2, -5, and -4, pointing out on a possible role of ILP6 as a mediator of the Aa5HT2B action.

Hemocytes transcriptome profile of the Chinese mitten crab (Eriocheir sinensis)

The Chinese mitten crab (Eriocheir sinensis) possesses an open circulatory system, in which hemolymph moves through interconnected sinuses or spaces surrounding organs called hemocoels. The hemocytes are classified into hyalinocytes, semigranulocytes, and granulocytes; and limited transcriptomics research is available. In this study, the transcriptome of the crab E. sinensis hemocytes was characterized. A total of 14,380,229 clean reads representing a total of 4.31 Gb nucleotides dataset were produced. A total of 67,047 contigs were obtained and 12.49% (8375) and 9.74% (6533) of the contigs were matched to data publicly available from the GenBank nr nucleotide and Uniprot databases, respectively. Among these contigs, 4344 contigs belong to three categories of Gene Ontology, 126 contigs to 21 subcategories of KEGG, and 4962 contigs to 25 categories of the COG database. A total of 508,379 and 345 transcripts of the E. sinensis showed >40% sequence similarity with transcripts expressed in the vertebrate blood cells from tilapia (Oreochromis niloticus), Chinese softshell turtle (Trionyx sinensis) and chicken (Gallus gallus) respectively. A total of 53,077 single nucleotide polymorphisms were identified and 9912 SSRs for microsatellite mining were found. The most frequent repeat motifs detected were dinucleotide repeats, accounting for 37.52% of the total repeats. Our data provides an important gene resource for the exploitation of molecular marker-assisted breeding, immune mechanisms and conservation of germplasm in the crab E. sinensis.

The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota

Public health research and vector control frequently require the rearing of large numbers of vector mosquitoes. All target vector mosquito species are anautogenous, meaning that females require vertebrate blood for egg production. Vertebrate blood, however, is costly, with a short shelf life. To overcome these constraints, we have developed SkitoSnack, an artificial blood meal replacement for the mosquito Aedes aegypti, the vector of dengue, Zika and chikungunya virus. SkitoSnack contains bovine serum albumin and hemoglobin as protein source as well as egg yolk and a bicarbonate buffer. SkitoSnack-raised females had comparable life history traits as blood-raised females. Mosquitoes reared from SkitoSnack-fed females had similar levels of infection and dissemination when orally challenged with dengue virus type 2 (DENV-2) and significantly lower infection with DENV-4. When SkitoSnack was used as a vehicle for DENV-2 delivery, blood-raised and SkitoSnack-raised females were equally susceptible. The midgut microbiota differed significantly between mosquitoes fed on SkitoSnack and mosquitoes fed on blood. By rearing 20 generations of Aedes exclusively on SkitoSnack, we have proven that this artificial diet can replace blood in mosquito mass rearing.

Identification of Uranotaenia sapphirina as a specialist of annelids broadens known mosquito host use patterns

Feeding upon vertebrate blood by mosquitoes permits transmission of diverse pathogens, including viruses, protozoa, and nematodes. Despite over a century of intensive study, no mosquito species is known to specialize on non-vertebrate hosts. Using molecular analyses and field observations, we provide the first evidence, to our knowledge, that a mosquito, Uranotaenia sapphirina, specializes on annelid hosts (earthworms and leeches) while its sympatric congener, Uranotaenia lowii, feeds only on anurans (frogs and toads). Our results demonstrate that Ur. sapphirina feeds on annelid hosts (100% of identified blood meals; n = 72; collected throughout Florida), findings that are supported by field observations of these mosquitoes feeding on Sparganophilus worms and freshwater leeches. These findings indicate that adult mosquitoes utilize a much broader range of host taxa than previously recognized, with implications for epidemiology and the evolution of host use patterns in mosquitoes.

Debugging diversity - a pan-continental exploration of the potential of terrestrial blood-feeding leeches as a vertebrate monitoring tool.

The use of environmental DNA (eDNA) has become an applicable noninvasive tool with which to obtain information about biodiversity. A subdiscipline of eDNA is iDNA (invertebrate-derived DNA), where genetic material ingested by invertebrates is used to characterize the biodiversity of the species that served as hosts. While promising, these techniques are still in their infancy, as they have only been explored on limited numbers of samples from only a single or a few different locations. In this study, we investigate the suitability of iDNA extracted from more than 3,000 haematophagous terrestrial leeches as a tool for detecting a wide range of terrestrial vertebrates across five different geographical regions on three different continents. These regions cover almost the full geographical range of haematophagous terrestrial leeches, thus representing all parts of the world where this method might apply. We identify host taxa through metabarcoding coupled with high-throughput sequencing on Illumina and IonTorrent sequencing platforms to decrease economic costs and workload and thereby make the approach attractive for practitioners in conservation management. We identified hosts in four different taxonomic vertebrate classes: mammals, birds, reptiles and amphibians, belonging to at least 42 different taxonomic families. We find that vertebrate blood ingested by haematophagous terrestrial leeches throughout their distribution is a viable source of DNA with which to examine a wide range of vertebrates. Thus, this study provides encouraging support for the potential of haematophagous terrestrial leeches as a tool for detecting and monitoring terrestrial vertebrate biodiversity.

The multiple roles of peroxiredoxins in tick blood feeding.

Hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) are generated through partial reduction of oxygen. The HO· are the most reactive and have a shorter half-life than H2O2, they are produced from comparatively stable H2O2 through Fenton reaction. Although controlling HO· is important and biologically advantageous for organisms, it may be difficult. Ticks are obligate hematophagous arthropods that need blood feeding for development. Ticks feed on vertebrate blood containing high levels of iron. Ticks also concentrate iron-containing host blood, leading to high levels of iron in ticks. Host-derived iron may react with oxygen in the tick body, resulting in high concentrations of H2O2. On the other hand, ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), to scavenge H2O2. Gene silencing of Prxs in ticks affects their blood feeding, oviposition, and H2O2 concentration. Therefore, Prxs could play important roles in ticks' blood feeding and oviposition through the regulation of the H2O2 concentration. This review discusses the current knowledge of Prxs in hard ticks. Tick Prxs are also multifunctional molecules related to antioxidants and immunity like other organisms. In addition, tick Prxs play a role in regulating the host immune response for ticks' survival in the host body. Tick Prx also can induce Th2 immune response in the host. Thus, this review would contribute to the further understanding of the tick's antioxidant responses during blood feeding and the search for a candidate target for tick control.

Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti

Aedes aegypti is a vector of medically important viruses including those causing Zika, dengue, and chikungunya. During mating, males transfer a number of proteins and other molecules to the female and these components of the male ejaculate are essential in shifting female post-mating behaviors in a number of insect species. Because these molecules are highly variable by species, and female post-mating behavior by species is also varied, behavioral assays testing the function of the ejaculate are necessary before we can develop control strategies targeting the mating system to reduce mosquito populations. Because increased survival in mosquitoes strongly increases vectorial capacity and can influence population sizes and potential risk we tested the effect of mating on female survival. The ejaculate can either promote or reduce female survival, as both have been shown in multiple insect species, yet this effect has not been directly assessed in mosquitoes. We compared survival of females in four treatment groups: mated females, virgin females, and virgin females injected with either an extract from the male reproductive glands or a saline control. Survival, blood feeding frequency, fecundity and cumulative net reproductive rate (R0) were determined after multiple feedings from a human host. Our results confirm that male reproductive gland substances increase female fecundity and blood feeding frequency, resulting in dramatic increases in fitness (R0). We also demonstrate, for the first time, an effect of male reproductive gland extracts alone on female survival, regardless of whether or not the female ingested a vertebrate blood meal. Thus, the effects of MAG extract on survival are not secondary effects from altered blood feeding. Collectively, we demonstrate a direct role for Ae. aegypti male-derived molecules on increasing female fitness, reproductive success and, ultimately, transmission potential for vector borne pathogens.

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes. One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti.

Mosquito-transmitted viral pathogens, such as dengue and Zika, afflict tens of thousands of people every year. These viruses are transmitted during the blood-feeding process that is required for mosquito reproduction, the most important vector being Aedes aegypti. While vertebrate blood is rich in protein, its high iron content is potentially toxic to mosquitoes. Although iron transport and sequestration are essential in the reproduction of vector mosquitoes, we discovered that culicine mosquitoes lack homologs of the common iron transporter NRAMP. Using a novel cell-based screen, we identified two ZIP and one ZnT genes as candidate iron transporters in the mosquito A. aegypti, the vector of dengue, Zika, and chikungunya. We determined the organ-specific expression pattern of these genes at critical time points in early reproduction. The result indicates modulation of these genes upon blood feeding, especially a ZIP13 homolog that is highly up-regulated after blood feeding, suggesting its importance in iron mobilization during blood digestion and reproduction. Gene silencing resulted in differential iron accumulation in the midgut and ovaries. This study sets a foundation for further investigation of iron transport and control strategies of this viral vector.

A new lipid carrier protein in the cattle tick Rhipicephalus microplus

Tick infestation in cattle reflects the main cause of economic loss to cattle producers. This is due to several reasons but mainly to their ability to feed on blood and generate a huge amount of eggs. Lipid transport in arthropods is achieved by highly specialized hemolymphatic lipoproteins, which resemble those described in vertebrate blood. Such lipoproteins continuously deliver lipids through the blood to growing eggs. The injection of radioactive [3H] palmitic acid into tick hemocoel showed that the gut, ovary, fat body and Gene’s organ were the main organs of incorporation of this labeled fatty acid. The rate of [3H] palmitic acid incorporation by the organs was high up to 30 min after injection. The [3H] palmitic acid incorporated by these organs was later found in phospholipids and neutral lipids. Here, we describe the purification and characterization of a key player of lipid dynamics in tick hemolymph. The Rhipicephalus microplus lipid-apolipoprotein complex (RmLCP) is a new high-density lipoprotein (1.18 g/mL), which accounts for over 90% of [3H] palmitic acid present in the hemolymph. It has a native molecular weight of 420 kDa and is composed of one subunit of 122 kDa. Protein identification analysis of RmLPC subunit showed two better hits: vitellogenin 2 (23% protein coverage) and vitellogenin 5 (29% protein coverage), respectively and similarities with hemolymphatic apolipoproteins of arachnids such as the tick Ixodes scapularis (80%), the mite Galendromus occidentalis (44%) and the spider Parasteatoda tepidariorum (43%) and also for the insects Locusta migratoria (45%), Drosophila melanogaster (42%) and Manduca sexta (47%) to vitellogenin 2 and tick Ixodes scapularis (83%), the crab Limulus polyphemus (55%) and the oyster Crassostrea gigas (55%) to vitellogenin 5. Furthermore, it shows a distinct lipid composition from most arthropod lipoproteins, being composed of 40% free cholesterol, 27% phospholipids, 20% triacylglycerol and 15% hydrocarbons. In addition to binding most hemolymphatic fatty acids, this lipoprotein also binds and transports free cholesterol. In conclusion, the present study provides insight into the macromolecules involved in arachnid metabolism, which have significant potential for future use for the biological control of ticks.